Preliminary investigation into the induction of
reproduction in Ulva spp. in Southeast Queensland for
mass cultivation purposes
Prue Pettett
B.Sc. (Environmental Science)
Grad. Dip. (Environmental Change Management)
Faculty of Science
University of the Sunshine Coast
January 2009
Submitted in partial fulfilment of the requirements for the degree of
Masters in Environmental Change Management
Statement of originality
I declare that this work does not contain any material which has been submitted by me previously
for any degree or diploma to any university, and to the best of my knowledge and belief, it does
not contain any material published or written by any other person, except where due reference is
made in the text.
Prue Pettett
January, 2009
2
Abstract
Ulva is a common component of marine intertidal flora in Australia with many species frequently
observed along the Queensland coastline. Three species of Ulva, U. lactuca, U. intestinalis and U.
prolifera were found to naturally occur at the Bribie Island Research Centre (BIRC) in Southeast
Queensland. Studies were undertaken to establish the most optimal conditions for growing Ulva in
the BIRC laboratory. These tests were conducted in order to condition the algal material prior to
the sporulation studies, offering more controlled material to assess treatment effects conclusively,
and helping eliminate other potentially confounding environmental factors. Results showed that a
stocking density of between 5-20 grams of Ulva per litre along with the addition of the soluble
fertiliser Aquasol at a rate of 87 mg/L of seawater was ideal for achieving a desired doubling of
growth per week.
In the wild the formation of Ulva fragments occurs naturally in the ocean through wave and storm
action. This breakage can trigger a survival response mechanism which stimulates the algae to
form and release gametes. By chopping the tissue, this process could be artificially simulated in
the laboratory and creating a simple and easy way to produce new individuals. Studies performed
into inducing sporulation in Ulva through a combination of fragmentation and renewal of medium
at BIRC showed that sporulation can be successfully induced in all three species of Ulva through
these methods, however it was found to be to a degree that would not meet the demands of
commercial production with on average a rate of only 33% achieved. While the current study did
not find a method suitable for a commercial application the results presented here contribute to
increasing our understanding about Ulva reproduction and set a platform for future work in to
cultivating Ulva within Southeast Queensland.
3
Acknowledgements
I express my sincere appreciation and gratitude to Dr Paul Palmer, Department of Primary
industries, for his valuable guidance and assistance in planning and designing of the
experimentation and for his improvements on the manuscript. I also thank Dr Peter Ducan,
University of the Sunshine Coast, for his intellectual stimulation and constructive criticism during
the writing of this manuscript. I thank Dr David Mayer, Department of Primary industries, for his
valuable assistance in the statistical analysis. I also extend a warm thanks to Dr Pia Winberg,
Sustainable Seafood Pty. Ltd, for her assistance in identification and to Dr John West, University
of Melbourne, and Dr Fred Gurgel, University of Adelaide, for providing answers with their
knowledgeable expertise in Ulva reproduction.
4
Table of Contents
Abstract..........................................................................................................................................3
Acknowledgements .......................................................................................................................4
List of Figures................................................................................................................................7
List of Tables .................................................................................................................................9
Chapter One: General Introduction ..............................................................................................10
1.1 Taxonomy and biology of Ulva ..........................................................................................10
1.1.1 Introduction................................................................................................................10
1.1.2 Assimilation of the genus Enteromorpha into Ulva ..................................................11
1.1.3 Reproductive biology.................................................................................................13
1.2 Commercial production of Ulva ..................................................................................15
1.2.1 Outline of the current industry...................................................................................15
1.2.2 Commercial uses and bioremediation potential.........................................................16
1.2.3 Seed cultures and future directions ............................................................................17
1.3 Inducing reproduction in Ulva ......................................................................................17
1.3.1 Light...........................................................................................................................17
1.3.2 Temperature ...............................................................................................................18
1.3.3 Changing the culture medium....................................................................................19
1.3.4 Dehydration................................................................................................................20
1.3.5 Thallus fragmentation ...............................................................................................20
1.3.6 Identifying and assessing sporulation ........................................................................21
Chapter Two: Identification of Ulva species. ...............................................................................24
2.1 Introduction.........................................................................................................................24
2.2 Materials and Methods........................................................................................................24
2.3 Results.................................................................................................................................25
2.3.1 U. lactuca...................................................................................................................25
2.3.2 U. intestinalis .............................................................................................................28
2.3.3 U. prolifera ................................................................................................................28
2.4 Discussion ...........................................................................................................................32
2.5 Conclusions.........................................................................................................................34
Chapter Three: Culture studies.....................................................................................................36
3.1 Introduction.........................................................................................................................36
3.2 Materials and Methods........................................................................................................36
3.2.1 Nutrient type and concentration.................................................................................37
3.2.2 Water type..................................................................................................................38
3.2.3 Species selection for fast growth ...............................................................................39
3.3 Results.................................................................................................................................40
3.3.1 Nutrient type and concentration.................................................................................40
3.3.2 Water type..................................................................................................................41
3.3.3 Species selection for fast growth ...............................................................................42
3.4 Discussion .....................................................................................................................44
3.5 Conclusions...................................................................................................................46
5
Chapter Four: Inducing sporulation in Ulva spp..........................................................................47
4.1 Introduction.........................................................................................................................47
4.2 Materials and Methods........................................................................................................48
4.3 Results.................................................................................................................................50
4.3.1 Sporulation.................................................................................................................50
4.3.2 Fouling .......................................................................................................................56
4.4 Discussion ...........................................................................................................................57
4.5 Conclusions.........................................................................................................................61
Chapter Five: Concluding summary .............................................................................................62
References......................................................................................................................................63
Appendix 1: Phytoplankton and other organisms observed in outdoor stock culture and
experimental Petri dishes ................................................................................................................69
6
List of Figures
Chapter One
Fig. 1.1 Schematic view of nuclear orientation and movement during somatic mitosis and
meiotic zoospore formation ...................................................................................................12
Fig. 1.2 Typical life cycle of Ulva; A: Male and female gametophyte, B: Biflagellate
gamete, C: Sporophyte, D: Quadriflagellate zoospore. 1N: Haploid; 2N: Diploid ...............14
Chapter Two
Fig. 2.1 U. lactuca in shallow drain (a), U. lactuca in deep mullet pond (b)........................26
Fig. 2.2 U. lactuca outdoor culture stock (a), U. lactuca thallus (b), Surface view of the
thallus cells (c), Transverse section of thallus cells (d) .........................................................27
Fig. 2.3 U. intestinalis outdoor culture stock (a), U. intestinalis thallus (b), Surface view
of the thallus cells (c), Transverse section of thallus cells (d) ...............................................30
Fig. 2.4 U. prolifera outdoor culture stock (a), U. prolifera thallus (b), Surface view of
the thallus cells (c), Transverse section of thallus cells (d) ...................................................31
Chapter Three
Fig. 3.1 Average cumulative weight of U. intestinalis under various nutrient
concentrations, mean ± se (n=3), different superscripts represent a significant difference
(P<0.05) .................................................................................................................................40
Fig. 3.2 Average cumulative weight of U. prolifera under various nutrient concentrations
and water type, mean ± se (n=3), different superscripts represent a significant difference
(P<0.05) .................................................................................................................................41
Fig. 3.3 U. prolifera not exposed to nutrients (a) and U. prolifera exposed to nutrients (b) 42
Fig. 3.4 Average cumulative weight of U. intestinalis, U. prolifera and U. lactuca with
Aquasol full, mean ± se (n=3), different superscripts represent a significant difference
(P<0.05) .................................................................................................................................43
Fig. 3.5 Average amount of grams put on each week by U. intestinalis, U. prolifera and
U. lactuca...............................................................................................................................43
Chapter Four
Fig. 4.1 Visual template designed in current experiment for ranking sporulation of Ulva
in Petri dishes.........................................................................................................................49
7
Fig. 4.2 Thallus in vegetative state (a), Thallus with displaced chloroplasts (b) Thallus
forming gametes (c) Thallus with fully formed gametes (d), Thallus after release of
gametes (e), gametes swimming in Petri Dish (f)..................................................................51
Fig. 4.3 Time profile of mean sporulation between species, mean ± se (n=12)....................53
Fig. 4.4 Time profile of mean sporulation between treatments, mean ± se (n=36)...............54
Fig. 4.5 Mean sporulation of fragmented and whole thalli in U. prolifera, U. lactuca and
U. intestinalis (n=84). ............................................................................................................54
Fig. 4.6 Time profile of mean sporulation between weeks, mean ± se (n=9) .......................55
Fig. 4.7 Water parameter values at the start of each week, mean (n=3)................................56
Fig. 4.8 Culture dish fouling (a) and outdoor culture stock fouling (b) ................................56
8
List of Tables
Chapter One
Table 1.1 Sporulation scale developed by Lersten and Voth (1960) ....................................22
Table 1.2 Sporulation scale developed by Nordby (1977)....................................................22
Chapter Four
Table 4.1 Ordered classification table used to assess sporulation in Ulva species ...............49
Table 4.2 Summary of repeated measures ANOVA with three grouping factors (Species,
Treatment, Week) and one trial factor (Time) and their interaction effects on mean degree
of sporulation in Ulva ............................................................................................................52
9
Chapter One
General Introduction
1.1 Taxonomy and biology of Ulva
1.1.1 Introduction
Ulva spp. (Chlorophyta, Ulvaceae) commonly known as sea lettuce, a member of the family
Ulvaceae are an early-successional algae quickly taking over new substrate after disturbances
(MBARI 2001). Ulva’s opportunistic success is attributed to their morphology which allows rapid
nutrient uptake, high capacity to store nutrients, and spontaneous mass reproduction (Santelices
and Ugarte 1987; MBARI 2001). These characteristics have allowed Ulva to develop a global
distribution with populations documented in Europe (Back 2000), North and South America
(Hoffmann and Ugarte 1985; Pringle 1986), Asia (Hiraoka et al. 2003), Pacific Islands (Smith
1947) and Australia (Phillips 1998).
Ulva occurs mainly in the littoral zone or near the high sub-littoral zone of the ocean foreshore. It
is extremely hardy and capable of existing in a wide range of salinities (2-35%). It often ascends
into estuaries, especially when there is an increase in nitrogen from pollution (Fritsch 1956;
Niesenbaum 1988). For much or all of its life Ulva is anchored via a disc shape holdfast to a
substrate which may include rocks, rope, woodwork or larger algae (Fritsch 1956; Pickett-Heaps
1975; Bold and Wynne 1985). Detachment regularly occurs through natural processes such as
storm or wave action or through human disturbances such as ship movements, but interestingly, as
10
long as it can access nutrients and light it can survive and grow whilst floating freely in the water
column.
1.1.2 Assimilation of the genus Enteromorpha into Ulva
Ulva was one of the first seaweed genera to be named and was recognised originally by Linnaeus
in 1753 as a single genus. The species was subsequently separated into two genera, namely Ulva
and Enteromorpha by Silva (1952), on the basis of the two significantly different morphologies.
The morphology of both Ulva and Enteromorpha characterised by a very thin undulating layer of
tissue which is essentially a bilayer of typical ulotrichalean cells containing chloroplasts and a
single to multiple pyrenoids (Figure 1.1). The cell axis is generally orientated at right angles to the
surface of the thallus with the chloroplasts mostly located on the outer side of the cell, however
depending on lighting conditions this can change with the chloroplasts able to move about freely
inside the cell (Fritsch 1956; Pickett-Heaps 1975).
It is this thin cellular layer that distinguishes the two genuses with Ulva consisting of a distromatic
blade (two cells thick) and Enteromorpha a tubular blade that is monostromatic (one cell thick)
(Bold and Wynne 1985).
However, several lines of evidence, found through molecular
investigations (Tan et al. 1999; Hayden et al. 2003; Shimada et al. 2003), and distribution and
bacterial studies (Nakanishi et al. 1996), suggest that these generic constructs are artificial and that
there is only one valid genus, Ulva.
11
Figure 1.1 Schematic view of nuclear orientation and movement during somatic mitosis and meiotic zoospore formation.
(From Braten & Nordby, 1973).
12
Molecular phylogenetic analyses of nuclear ribosomal RNA ITS sequences by Tan et al. (1999)
indicated that the two genera Ulva and Enteromorpha should not be separated. Analyses showed
that the radical change in gross morphology of the two genera has arisen independently several
times through their evolutionary history, suggesting that this morphological flexibility is the result
of some form of developmental switch resulting in either blades or tubes. This was supported by
further studies conducted on the nuclear ribosomal internal transcribed spacer DNA (ITS nrDNA)
(Hayden et al. 2003) and ITS and rbcL in Ulva and Enteromorpha species (Shimada et al. 2003),
again confirming that Ulva and Enteromorpha are not separate genera.
Bacterial studies by Nakanishi et al. (1996) found that Ulva pertusa lost its typical morphology
when cultured in a synthetic media that lacked symbiotic bacteria. It was also found that with the
reinfection of certain bacteria that this species can regain its typical foliaceous or tubular
morphology. These authors concluded that the direct contact between Ulva and the bacterial strain
was necessary for normal morphological growth.
Since Ulva is the older genus, there is growing consensus that Enteromorpha should be reduced to
the synonymy of Ulva (Nakanishi et al. 1996; Shimada et al. 2003). In the following report
therefore only the single genus Ulva will be recognised.
1.1.3 Reproductive biology
The reproductive cycle of Ulva is known as an alternation of generations (Dawson 1956). It
consists of two phases (see Figure 1.2) a haploid (one set of chromosomes) gametophyte stage
which produces biflagellate gametes and a diploid (two sets of chromosomes) sporophyte stage
which produces quadriflagellate zoospores (Trainor 1978). These two sequential generations are
13
almost identical in appearance (isomorphic); the only differences being the number of flagella in
the motile single celled stages, and the cell sizes with haploid cells distinctly smaller than diploid
cells (Nordby 1974; Pickett-Heaps 1975; Trainor 1978).
Figure 1.2 Typical life cycle of Ulva; A: Male and female gametophyte, B: Biflagellate gamete, C: Sporophyte, D:
Quadriflagellate zoospore. 1N: Haploid; 2N: Diploid (from Beach et al. 1995).
In the wild the release of zoosporangia or gametangia collectively known as swarmers has been
observed to occur around spring tides; however this may vary between species (Niesenbaun 1988).
Studies conducted by Pringle (1986) concluded that U. intestinalis swarmer discharge is not
synchronised with the occurrence of spring tides, but rather by the lunar cycle. Reproductive
formation due to the lunar cycle was also recorded by Christie and Evans (1962) who observed
that swarmer discharge in U. intestinalis peaks 3-4 days prior to the highest tides on each lunar
cycle.
The lunar cycle was also found to play an important role in the laboratory. U.
pseudocurvata under laboratory conditions was found to have 7-day reproductive peaks, however
after the introduction of artificial moon light every four weeks the reproductive rhythm became
more synchronised with only a monthly release (Luning et al. 2008).
14
Ulva’s life-cycle occurs without the formation of special generative organs. Nevertheless,
reproductive material can at times significantly dominate the biomass of these algae e.g. it can be
up to 60% of the total biomass in species such as U. lactuca (Nordby and Hoxmark 1972;
Niesenbaum 1988). In some cases this mass reproduction can have detrimental consequences for
the environment.
In shallow coastal eutrophic lagoons or estuaries, primary production from Ulva can become so
high that subsequent decomposition processes can lead to oxygen depletion (Sfrisco et al. 1992).
This effect is most pronounced in the bottom water or sediment which then causes anoxia and
hydrogen sulphide production. These conditions then cause detrimental effects on the thalli
themselves with reports that exposure to anoxia and sulphide gradually reduces the growth
capacity of U. lactuca (Giordani et al. 1997)
1.2 Commercial production of Ulva
1.2.1 Outline of the current industry
The commercial farming of macroalgae (seaweed) has a long history dating back hundreds of
years. Currently there are approximately 200 species of macroalgae used worldwide, of which
around 10 are intensively cultivated (Zemke-White and Ohno 1999; Wikfors and Ohno 2001).
Macroalgae global utilization is on the increase with annual harvests worth around $5-6 billion U.S
per year (Wikfors and Ohno 2001; Luning and Pang 2003), making it among the most important
cultivated marine organisms. Wet annual harvest weights equate to approximately six million
15
metric tonnes, and 90% of this is produced via culture-based practices in China, Korea, Japan,
Philippines, Indonesia, Chile, Taiwan, Vietnam, Russia and Italy (FAO 2001; NAAS 2003).
1.2.2 Commercial uses and bioremediation potential
Seaweeds are a staple part of the diet of many people living in countries such as Japan, China and
Korea. In particular, species such as Nori (Porphyra spp.), Wakame (Undaria pinnatifida) and
Kombu (Laminaria japonica) are used as ingredients in soups, salads and noodles, or are served
with meat and/or fish as a vegetable (Radmer 1996). Seaweed extracts are also used in a very wide
range of products including soaps, shampoos, cosmetics, toothpaste, textiles, sanitary napkins, air
fresheners, paper products, culture media, fungicides, beer, meat, ice-creams, syrups and salad
dressings (NAAS 2003).
More recently seaweeds are becoming used as a cheap and effective biofilter. Land and oceanbased mariculture creates polluting waste nutrients which consist primarily of either the metabolic
waste of the cultured species or broken down uneaten food (Naylor et al. 2000). Macroalgae are
well known to be able to partly restore water quality and reduce the environmental impact derived
from high nutrient loads (Neori et al. 2004). They have been successfully used as biofilters for
numerous aquaculture species including abalone (Neori et al. 2000), shrimp (Jones et al. 2002),
fish (Neori et al. 1996) and sea cucumbers (Wang et al. 2007).
16
1.2.3 Seed cultures and future directions
Traditionally, macroalgae species for mariculture have been obtained through what is known as
“natural seeding”. This method involves placing nets, ropes and other spore settlement devices into
wild populations during early autumn and spring when the algae are naturally sporulating.
However this is becoming less feasible as wild stocks in many countries have been depleted by
overharvesting and excessive pollution (Niesenbaum 1988; NAAS 2003). Other reasons resulting
in a switch from natural seeding to artificial reproductive induction include minimising variability
in production levels by reducing genetic diversity and reducing seasonal effects by allowing
reproduction to occur at almost any time of the year when supplied with sufficient parental
biomass (Niesenbaum 1988; Ohno 1993; Dan 2002).
1.3 Inducing reproduction in Ulva
Vegetative cells can transform directly into zoosporangia or gametangia at any time, and this
reportedly can be stimulated through a number of environmental shock treatments including
changes in photoperiod, temperature, salinity, nutrients, pH, drying and through fragmentation
(Pickett-Heaps 1975; Hiraoka and Enotomo 1998).
1.3.1 Light
As the release of reproductive cells is dependent on photosynthesis, light is an important factor in
the spawning of Ulva, with a positive correlation between availability of light in the water column
and spore biomass. It is believed that the increase in light along with other environmental factors
is responsible for the induction of reproductive material in spring and summer (Sousa et al. 2007).
17
This idea was also previously noted by Smith (1947) who believed that sporulation in Ulva species
was related to periodic increases of light exposure.
The light requirements for maximal spore release in U. pertusa were found by Han et al. (2008) to
be >30 μmol/m2. However, Dan et al. (2002) found that Ulva could still reproduce reasonably well
at lower light levels, in the order of 16 μmol/m2. The minimum light requirements for reproduction
have been found to be much lower at between 5 and 10 μmol/m2 (Dan et al. 2002; Han et al.
2003).
1.3.2 Temperature
Ulva is extremely tolerant to temperature changes with freezing experiments conducted by
Kamermans et al. (1998) demonstrating that the algae are able to survive at -5ºC for 2 weeks when
kept in the dark. This refers only to plant tissue, since sexual reproduction at this temperature is
negligible.
Maximal sporulation percentages occurred between 15 and 20°C in U. pertusa (Han et al. 2005).
There were significant declines at or below 10°C and at or above 25°C and that at 5°C no
sporulation occurred. Earlier work showed that the optimum temperature for sporulation of U.
mutabilis is 21-22°C (Nordby 1977). However, Nordby also concluded that temperature for
sporulation is by no means absolute if given enough time to sporulate. When U. mutabilis
fragments were incubated for 10 days, high degrees of sporulation (>50%) could occur in
temperatures as high as 28-29°C and as low as 15°C.
18
Rapid changes in temperature can also induce reproduction in Ulva. Washing U. lactuca in filtered
seawater at 2°C and then returning it to a similar medium at 22°C caused all of the algal material
to become reproductively active within 18 hours (Niesenbaum 1988).
1.3.3 Changing the culture medium
Renewal of the suspension medium has also been shown to induce generative division in
vegetative cells. This is reported to be well accepted and common practice in laboratories wanting
to induce reproduction in Ulva (Nilsen and Nordby 1975). Increasing proportions of freshly added
medium, the amount of sporulation also increased (Thaidens and Zeuthen 1967). This is believed
to be caused by the associated changes in salinity and pH that stimulate the formation and release
of zooids.
Salinity can influence spore release by affecting the turgor pressure and diameter of the exit pore
of sporangia (Han et al. 2008). Spore germination and growth is adversely affected by low
salinities; growth is significantly reduced at salinities below 5 ppt (Sousa et al. 2007). Growth was
also found to be highest at levels above 20 ppt, and with 35 ppt the most effective. Other species
have also shown a tendency for optimal spore release rates at oceanic salinities (35 ppt). For
example, Han et al. (2008) found that maximal spore release for U. pertusa occurred between 25
and 35 ppt.
The pH of the suspension medium has also been shown to affect sporulation in at least two Ulva
species. The optimum pH for inducing sporulation in U. mutabilis is thought to be 8.0-8.5 (Nordby
1977), and the optimum pH for spore release in U. pertusa is reportedly between 7 and 9 (Han et
al. 2008).
19
1.3.4 Dehydration
Since Ulva occurs naturally in the tidal zones of oceans, it is periodically subjected to varying
levels of dehydration. Sporulation could be induced by dehydration in U. lobata, U. taeniata, U.
linza, U. angusta and U. stenophylla (Smith 1947). The procedure involved removing thalli from
the water for an hour and then re-immersing them. When treated in this manner most of the blades
reportedly released swarmers within 5-10 minutes. However, it is probable that in this case the
first phase of reproduction had already been completed and that the algae just needed another
stimulus to complete the second phase of reproduction.
Corradi et al. (2006) induced
gametogensis in Ulva by dehydrating the thallus on a filter paper at 20 ± 1°C and 25% humidity
for 10, 20, 30 and 40 minutes in a climate-controlled chamber. In contrast with Smith (1947) thalli
subjected to dehydration took three days to produce gametes; presumably in this case it was not
already reproductively mature.
1.3.5 Thallus fragmentation
Fragmentation has been found to be a highly successful form of treatment to induce sporulation
and has been used to stimulate the release of zooids in Ulva for many years (Nordby 1974; Dan et
al. 2002). Fragmentation can greatly improve sporulation rates with studies finding that tearing of
the thallus can increase sporulation from 15.8 to 80% (Nordby 1977). The cutting of the thalli has
also been found to improve synchronisation. Research by Nordby and Hoxmark (1972) reported a
high degree of synchronous zooid formation by cutting the thallus of U. mutabilis with razor
blades into small fragments of around 0.4 mm x natural width. However this contradicted previous
findings by Lersten and Voth (1960) who found no significant differences in zooid discharge
between fragmented and non fragment thalli. However, Lersten and Voth used comparatively
20
larger thalli fragments (4 mm) than used in other studies, and this may explain their differing
results.
Studies by Hiraoka and Enomoto (1998) into fragment size found that there were significant
differences in the degree of sporulation between fragment sizes. They concluded that zooid
formation increases as fragment size decreases. Nordby (1977) found that fragments that are too
small should be avoided since the edge cells tend to sporulate later than the inner cells.
Various methods and instruments have been used to experimentally fragment the thalli including
tearing the thalli with needles (Nordby 1977), with a razor-blade blender (Nordby and Homark
1972), with a leather craft punch (Dan et al. 2002; Hiraoka 2003; Hiraoka and Oka 2008) or a
double-bladed ophthalmic knife (Lersten and Voth 1960), as well as razor blades (Verlaque et al.
2002) and herb choppers (Stratmann et al. 1996). Using a leather craft punch is now becoming a
more favoured experimental method since the circular shape creates more surface area exposed to
fragmentation, however there are no documented comparisons of the effectiveness of various
instruments, and there appears to be no commercially applicable method that is suitable for large
volume treatments.
1.3.6 Identifying and assessing sporulation
Reproductive maturation usually occurs within two to three days of applied stimuli, and often
between the hours of 11:00 pm and 4:00 am. Swarmer discharge occurs the same day, generally a
few minutes after sunrise (Carter 1926; Hiraoka and Oka 2008). The sex of biflagellate gametes
can be distinguished from each other by their colour; female gametes appear as a dark green mass
while male gametes appear more yellow green in colour due to their prominent eyespot.
21
On a broad scale, sporulation can be identified in Ulva thalli through a change in colouration.
During the vegetative state the thallus is a dark or slightly yellow green. However, during
reproductive maturation, the thallus turns to a dark olive to orange-brown (Hiraoka and Enomoto
1998; Han and Choi 2005).
When the zooids are released the thallus becomes colourless in contrast to green non-discharged
sections, and this presents an opportunity to rate levels of sporulation by eye in a qualitative
fashion. Various sporulation scales have been developed to assist in gauging the level of
sporulation through assessing the degree of change in colour of the thallus. Lersten and Voth
(1960) developed the following simple scale based on how much green colour remained in the
thallus (Table 1.1):
Table 1.1 Sporulation Scale developed by Lersten and Voth (1960).
Discharge level
No discharge
Discharge less than one-third of the thallus
Discharge from one-third of the thallus
Discharge from more than two-thirds of the thallus
Value
0
1
2
3
Nordby (1977) taking a similar approach however developed five categories which were weighted
for sporulation as follows (Table 1.2):
Table 1.2 Sporulation Scale developed by Nordby (1977).
Discharge level
0% discharged
>10% discharged
10-50% discharged
50-90% discharged
90-100% discharged
Value
0
0.5
3
7
10
22
Sporulation can also be measured through quantitative measurements including measuring the
density of the zooid with a haemocytometer and by measuring the chlorophyll a concentration of
the suspending medium (Niesenbaum 1988; Hiraoka and Oka 2008).
The objectives of the following study were to; identify all Ulva species naturally occurring at
BIRC (i), establish the most optimum conditions for growing Ulva in the laboratory (ii), test if
Ulva can be successfully induced into sporulation through a combination of fragmentation and
renewal of medium and if so quantify the degree of sporulation and assess if it is suitable for a
commercial application.
23
Chapter Two
Identification of Ulva species
2.1 Introduction
Ulva is a common component of marine intertidal flora in Australia with many species frequently
observed along the Queensland coastline (Cribb 1956). An extensive literature search has found
that there is good documentation of species diversity within Queensland with the following species
of Ulva recorded to occur; U. ahlneriana, U. clathrata, U. compressa, U. fasciata, U. flexuosa, U.
gunniana, U. intestinalis, U. lactuca, U. linza, U. paradox, U. procera, U. prolifera, U. ralfsii, U.
reticulata and U. rigida, however there is very limited information available about the exact
locations and seasonal abundance of this genus within Queensland (Cribb 1956: Cribb 1996;
Phillips 1997; Davie 1998; Phillips 2007).
Natural populations of Ulva were found to regularly occur in various places at the Bribie Island
Research Centre (BIRC) in Southeast Queensland, however their taxonomy was uncertain. The
following study sought to identify all Ulva species occurring at BIRC and document their
morphological characteristics and habitat for future reference. This was seen as a necessary
starting point for using this source of Ulva species in sporulation induction trials.
2.2 Materials and Methods
Thalli of Ulva were collected from outside drains and ponds at BIRC in April 2008. The thalli
were collected and photographed immediately with the use of a digital camera and light
microscope (Nikon Microphot-FXA), at various levels from broad scale views to microscopic.
24
Transverse sections were taken by holding the thallus down with a coverslip and finely slicing the
thalli with a razor blade; this was later photographed with the use of a light microscope. Cellular
and thallus measurements were taken with the use of a ruler and micrometer. These photographic
records, cellular measurements, and thalli measurements along with further live samples were used
to sequentially identify the species present at that time.
Identification was conducted in
collaboration with Dr Pia Winberg, director of Sustainable Seafood Pty. Ltd who had prior
experience with Ulva identifications. Generally, species are primarily distinguished based on
anatomical grounds using the dimensions, length and breath ratios and lateral profiles observed in
transverse sections, including blade morphology such as branching (Phillips 1988). Identification
was assisted with use of the following guides: Cribb (1956), Phillips (1988), Cribb (1996) and
Kraft (2007). Habitat descriptions were noted and abundance was visually observed and recorded
over a seven month period (April to October).
2.3 Results
The study of the BIRC facility established that three species of Ulva are commonly present. These
species were identified as U. intestinalis, U. prolifera and U. lactuca. Their habitat and biology
descriptions were made as follows:
2.3.1 U. lactuca
U. lactuca was found growing both in shallow drains with less than 2 cms of water and in 1-2 m
deep ponds. U. lactuca was noted to be growing under the following conditions: full sun; slow to
still water movement in ponds; fast and strong water movement in drains; natural seawater salinity
with higher than natural nutrients levels due to nutrient supply from aquaculture ponds. The
25
morphological structure was seen to change with different water depths, with a more crumpled and
compact form in shallow areas and a broader more flat form in deeper waters (Figures 2.1 and 2.2).
In general it can be described as a thin flat green alga with broad and crumpled thalli 18 cms or
more in length and up to 30 cm wide. There were significant variations in the size of this species
detected, with the largest specimen observed being 77 cms long and 33 cms wide. The alga was
found both growing attached via a small disc-shaped holdfast and also unattached floating on the
surface.
The thallus is generally translucent and green to dark green in colour, however yellowing occurs
during times when there are only limited nutrients. Thalli were also found to bleach white when
exposed to air for extended periods of time.
The thallus in U. lactuca is distromatic (two cells thick) with each of the cells containing one to
three pyrenoids. Cells are irregularly arranged, cell diameter in surface view is 10-15 μm, and
thallus thickness is 80 μm. Abundance of this species was found to be at highest during the
months of August, September and October.
a
b
Fig. 2.1 U. lactuca in shallow drain (a), U. lactuca in deep mullet pond (b).
26
a
b
15 cms
c
d
10 µm
10 µm
Fig. 2.2 U. lactuca outdoor culture stock (a), U. lactuca thallus (b), Surface view of the thallus cells (c),
Transverse section of thallus cells (d).
27
2.3.2 U. intestinalis
U. intestinalis was found growing both attached (via a disc-like holdfast) and unattached in
shallow drains (>2 cms) and deep ponds (1-2 m) in troughs at BIRC (Figure 2.3). It was noted to
be growing in the following conditions: full sun; slow to still water movement; natural seawater
salinity with high nutrients levels from aquaculture ponds. It was observed to form thick mats (up
to 5 cm thick) which floated on the water surface when grown in deep ponds.
U. intestinalis can be described as having a thin ruffled unbranched fronds 1 cm wide and up to 20
cms or more in length. The shape is variable with some widening in the mid region. Thalli are
bright green in colour with yellowing occurring when exposed to a lack of nutrients.
The thallus is monostromatic with one to four pyreniods per cells. Cell diameter in surface view is
10 μm, thallus thickness is 30 μm. U. intestinalis was found to be highest in abundance at BIRC
during the months of April, May and June.
2.3.1 U. prolifera
U. prolifera has thin hollow tubular monostromatic thalli which are bright green to dark green in
colour (Figure 2.4). Again, yellowing is observed to occur when there is a lack of nutrients. It was
found both attached via a disc-like holdfast and unattached in shallow drains (>2 cms) and deep
ponds (1-2 m). The thalli were found exposed to full sun, natural seawater salinity, with nutrient
supply from aquaculture ponds and slow bubbling water movement.
28
The thallus is 1 mm wide and 10-15 cms in length, cell diameter in surface view is 10 μm, thallus
thickness is 30 μm. Abundance of U. prolifera was found to be highest at BIRC during the months
of June, July, August, September and October.
29
a
b
5cms
c
d
10µm
10µm
Fig. 2.3 U. intestinalis outdoor culture stock (a), U. intestinalis thallus (b), Surface view of the thallus cells (c), Transverse
section of thallus cells (d).
30
a
b
c
d
10µm
Fig. 2.4 U. prolifera outdoor culture stock (a), U. prolifera thallus (b),Surface view of the thallus cells (c), Transverse section of
thallus cells (d).
31
2.4 Discussion
The three species identified at BIRC in this study, U. lactuca, U. prolifera and U. intestinalis, have
been reported from this part of Queensland by several previous authors (eg: Cribb 1956: Cribb
1996; Phillips 1997; Phillips 2007). Hence, there is reasonable confidence that the species
identifications in this study are correct. Nevertheless, it should be recognised that some uncertainty
in this regard still exists. Ulva presents extreme difficulties in taxonomy due to its wide
intraspecific variations in morphologies. Morphological differences between species are notably
small and difficult to detect (Bliding 1963). While it was easy to discriminate between the species
classified in this study as U. lactuca, U. intestinalis and U. prolifera, because they each have very
distinct growth forms, it is generally not so easy to distinguish these species from other very
similar species within this genus.
Species such as U. lactuca have been noted to resemble other species, such as U. linza, and it
seems likely that within Australia these species have been frequently confused (Phillips and
Clayton 1983). Another set of species which have been misidentified are U. intestinalis and U.
compressa. These two species are morphologically so similar that they are almost
indistinguishable. One morphological characteristic used to separate these two species is the
degree of branching; U. intestinalis is generally unbranched but U. compressa takes on a branched
morphology. However under different environmental conditions this branching tendency can vary.
In brackish water conditions U. intestinalis can become branched and U. compressa can revert to
an unbranched form, further complicating attempts at morphological discrimination (Tan et al.
1999). This ability for Ulva to switch morphological characteristics under different environmental
conditions makes Ulva taxonomy difficult.
32
The present study recognised the need to document habitat descriptions when identifying the
species. In the current study significant morphological changes were observed in U. lactuca under
different environmental conditions. When grown in a deep still pond the thalli were flat and
smooth however in shallow high water flow drains the thalli were compact and ruffled (see Fig
2.1). This is believed to have occurred due to the changes in water movement. Similar gross
changes in size and morphology in Ulva have been observed in other studies. Mshigeni and
Kajumulo (1979) found U. fasciata to change morphology when exposed to different water
conditions with thalli becoming smooth and long when placed in calm-water and more compact
and dwarf like when placed in wave-exposed water.
The identification techniques employed in the current study are considered to be a scientifically
acceptable form of identification (Phillips 1988); however more recently, nuclear ribosomal DNA
internal transcribed spacer (ITS) sequences have been used for the identification of Ulva species
(Dion et al. 1998). These rDNA data have been accumulated and can be utilised for comparison
among difficult to identify Ulva species, and of course they are much more accurate for
identification (Blomster et al. 1998; Shimada 2003). This was however not feasible in the current
project due to limited resources.
Ulva is known as a pseudo-perennial with most of the thalli dying back during warmer months
however the holdfast portions are perennial and new thalli are then regenerated through this basal
material during cooler times of the year (White and Keleshian 1994). An extensive literature
search found very little documentation of seasonal changes with most studies not going any further
than stating that Ulva abundance is highest during the cooler months (eg: Cribb 1996). Outdoor
Ulva assemblages at BIRC were seen to fluctuate significantly between months. This is yet to be
fully investigated but could possibly be due to a number of factors including changes in nutrient
input, growth, salinity, temperature, light, grazing pressure from invertebrates, sporulation and
33
decomposition which have all being previously found to change Ulva biomass (Sfriso &
Marcomini 1996; Balducci et al. 2001). This fluctuation in Ulva assemblages was not viewed by
scientists at BIRC as a significant problem for its use as a bioremediation agent, since one useful
species seems to give way to another in a timely and uninterrupted fashion. In other words, when
one species of Ulva sp. died off another increased in occurrence, filling the niche space and
utilising the available light and nutrient resources. Therefore, its nutrient stripping abilities were
maintained as long as a reasonable biomass of at least one form was maintained.
The abundance of Ulva at BIRC may not be representative of wild populations under unpolluted
natural conditions, because Ulva in certain localities at the research station is under more
favourable conditions having higher nutrient supplies, stable water levels with no tidal effects or
storm movement, and metered control over herbivorous grazers. In order to better understand wild
populations of Ulva in Southeast Queensland further studies would need to be broadly undertaken
in its many suited habitats along the Queensland coastline.
2.5 Conclusions
Three species of Ulva were found to occur naturally in drains and ponds at BIRC. The species
were identified as U. lactuca, U. intestinalis and U. prolifera. Whilst there is reasonable
confidence that the species were identified correctly, some uncertainty still remains due to the
extreme difficulties in Ulva taxonomy. Molecular analysis would need to be undertaken in order to
obtain complete certainty however this was not feasible in the current study. Seasonal variations
in outdoor Ulva populations were observed to occur. The reason behind the increases and
decreases in biomass is yet to be investigated, however some possibilities include changes in
growth, nutrients, salinity, temperature, light, grazing pressure from invertebrates, sporulation and
decomposition. Further studies need to be undertaken over several full years in order to better
34
understand the population dynamics of U. prolifera, U. lactuca and U. intestinalis at BIRC.
Observations of Ulva abundance at BIRC may not reflect wild populations because Ulva in the
outdoor water system at BIRC can at times be presented with very favourable growth conditions.
Further studies would need to be undertaken to better describe the patterns of growth and to assess
the true abundance of Ulva in the wild within Southeast Queensland.
35
Chapter Three
Culture Studies
3.1 Introduction
Ulva is a hardy alga that is highly tolerant of variable salinity, temperature and water quality. It
grows rapidly in nutrient-rich habitats and is often used as a bioindicator of organic and inorganic
pollution in waterways (Fletcher 1996; Leal et al. 1997). Ulva is so successful at absorbing
nutrients that it can be integrated into land-based aquaculture practices and used as a biofilter
partly restoring the water quality and reducing the environmental impact derived from the high
nutrient loads (Neori 1996; Neori et al. 1996; Neori et al. 2004).
Ulva is currently growing successfully in outdoor troughs and drains at BIRC, however in order to
undertake sporulation studies the alga must first be successfully grown in the laboratory. The
following chapter aimed to establish the optimal conditions for growing Ulva in the BIRC
laboratory. This would allow it to be conditioned prior to sporulation studies, offer more controlled
material to assess treatment effects conclusively, and help eliminate other potentially confounding
environmental factors.
3.2 Materials and Methods
Three different experiments were conducted as a part of optimising culturing conditions in the
laboratory. They focussed on: (1) optimising available nutrient types and concentrations; (2)
investigating the effect of different source waters; and (3) assessing comparative growth rates of
the various species to identify the fastest grower under the experimental conditions.
36
3.2.1 Nutrient type and concentration
U. intestinalis was gathered from outdoor stock cultures at BIRC and washed in fresh water three
times to kill potential grazers (eg: amphipods). These wild gatherings were divided into 10 g lots
(wet weight). Each of these 10 g lots was then placed into a 3 L clear culturing bowl containing 2
L of filtered (1 µm) seawater and one of the following five treatments. Nutrient concentrations
were based on a desired doubling of growth per week which would require an assumed 40 mg of
nitrogen per week:
•
No added nutrients (seawater control)
•
Full strength Aquasol soluble fertiliser - 87 mg per litre
•
Half strength Aquasol soluble fertiliser - 43.5 mg per litre
•
Full strength Nanno mass culture mix - 199 mg per litre
•
Half strength Nanno mass culture mix - 99.5 mg per litre
The Aquasol fertiliser used is a commercially available product which provides a balanced nutrient
mix containing the full range of trace elements necessary for plant growth. It is easily dissolved in
seawater and is routinely used at BIRC to grow the marine microalgae Isochrysis galbana. The
Nanno mass culture mix is a mixture of agricultural grade fertilisers that are used to grow mass
cultures of the marine microalgae Nannochloropsis oculata. It consists of ammonium sulphate,
superphosphate and urea in the w/w ratio of 15.3: 1.4: 1.0 (Palmer et al. 2007).
Five replicates of each of these treatments were made giving a total of 25 experimental units. Each
bowl was randomly placed in a light cupboard using a random table of numbers. Treatments were
cultured under laboratory conditions at ambient temperature (approximately 20˚C) and exposed to
37
a white light at 10,000 lux under a 12 hour light: 12 hour dark regime for two weeks. Weight
measurements were recorded at the same time every week by shaking the algae dry in a fish net
and then weighing on an electronic scale (0.01 g accuracy).
3.2.2 Water type
U. prolifera material was collected from outside stock cultures at BIRC and washed in fresh water
three times. Drainage water was collected weekly from a cultured-marine-worm sand bed and
passed through a filter to 1 µm prior to use. Seawater used in the trial was also filtered to 1 µm. 10
grams of U. prolifera was placed into each 3 L clear bowl with 2 L of either water type and one of
the following treatments was applied:
ƒ
Seawater - full Aquasol - 87 mg per litre
ƒ
Seawater - no nutrients
ƒ
Worm bed water - full Aquasol - 87 mg per litre
ƒ
Worm bed water - no nutrients
Five replicates were made for each of the treatments making a total of 20 experimental units. Each
bowl was placed in a light cupboard (10,000 lux) on a 12hr light: 12 hr dark cycle at ambient
temperature (approx 20˚C) for three weeks. At the same time every week wet weight
measurements were taken by shaking the algae dry in a net and then weighing on electronic scales.
38
3.2.3 Species selection for fast growth
U. prolifera, U. lactuca and U. intestinalis were collected from outdoor stock cultures at BIRC.
The algal material was then washed in fresh water 3 times and divided into nine 10 g lots. Each of
the 10 g lots were then placed in a 3 L clear bowl with 2 L of nutrient enriched (Aquasol full
strength; 87 mg/L) seawater. The bowls were randomly placed onto a wire rack in a light
cupboard on a 12 hr light: 12 hr dark cycle at ambient temperature (approx 20˚C) for two weeks.
Once a week the wet weight of algae (shaken dry as above) was recorded for each of the
experimental units.
Data Analysis
In the nutrient type and concentration experiment a one-way ANOVA and Tukey’s HSD post hoc
test were performed with the use of the statistical package SPSS (14.0) to assess if there were
significant differences between the five nutrient types. In the water type experiment a one-way
ANOVA was performed to test for the effects of water type on growth of U. prolifera (P=0.05). If
the ANOVA was significant, a Tukey’s HSD test was applied to test for significant differences
within the factor levels (P=0.05). For the species selection experiment a one-way ANOVA and
Tukey’s post-hoc test was be performed to assess the significance of differences between the
growth of U. intestinalis, U. lactuca and U. prolifera.
39
3.3 Results
3.3.1 Nutrient type and concentration
Aquasol full concentration experienced the highest quantity of growth throughout the experiment
with a gain of 22.65 grams within two weeks (Figure 3.1). This fertiliser treatment produced
significantly higher weight gains than all other treatments except the Aquasol half treatment
(which gained 18.47 grams in two weeks). Nanno full performed the worst in terms of growth with
only 5.80 grams gained in two weeks. A one-way ANOVA determined that there was a significant
difference in growth between nutrient types and nutrient concentrations (P<0.05).
Post-hoc analysis (Tukey’s test) determined that the difference is located between the control and
Aquasol full (P<0.001), Aquasol full and both Nanno full (P<0.001) and Nanno half (P<0.05) and
finally between Aquasol half and Nanno half (P<0.05) (Fig 3.1).
40
c
Weight (grams)
35
b,c
30
a,b
a
25
20
a
Nano Full
Control
Nano Half
15
Aquasol Half
10
Aquasol Full
5
0
Initital
Week 1
Week 2
Time
Fig. 3.1 Average cumulative weight of U. intestinalis under various nutrient concentrations,
mean ± se (n=3), different superscripts represent a significant difference (P<0.05).
40
3.3.2 Water type
Treatments applied in the water type experiment were shown to significantly effect the results
(P<0.001) (Figure 3.2). Post-hoc analysis (Tukey’s test) determined that the addition of Aquasol
significantly increased algal weight gains however there were no statistically significant
differences observed between water types (P>0.05).
b
50
b
45
Weight (grams)
40
35
a
30
Wormbed Control
a
Seawater Control
25
Wormbed Full Aquasol
20
Seawater Full Aquasol
15
10
5
0
Intitial
Initial
Week
Week
1 1
Week
Week
2 2
Week
Week 3 3
Time
Time
Fig. 3.2 Average cumulative weight of U. prolifera under various nutrient concentrations and water type, mean ± se
(n=3), different superscripts represent a significant difference (P<0.05)
41
Through visual observation it was found that nutrient availability changed the colour of the thallus.
Ulva thalli exposed to no-nutrients treatments became pale green to yellow in colour whereas Ulva
thalli treated with a strong nutrient concentration turned dark green (Figure 3.3).
a
b
Fig. 3.3 U. prolifera not exposed to nutrients (a) and U. prolifera exposed to nutrients (b).
3.3.3 Species selection for fast growth
All three species experienced a high degree of growth during the two weeks of experimentation. U.
lactuca demonstrated the highest weight gains with an increase in wet weight of 71.7%. It was
closely matched by U. prolifera with a 70.7% increase in wet weight. The least amount of growth
was seen in U. intestinalis with a final average weight of 12.84 grams or an increase of 28.4%. A
one way ANOVA performed showed that there was a significant difference in growth between
species (P<0.05) (Figure 3.4).
42
Post hoc analysis (Tukey’s test) established that the weight gains of U. prolifera and U. lactuca
were not significantly different (P>0.05). However there was a significant difference between U.
intestinalis and U. prolifera (P<0.05) and between U. intestinalis and U. lactuca (P<0.05).
b
20
Weight (grams)
18
16
b
a
14
U. intestinalis
12
10
U. prolifera
8
U. lactuca
6
4
2
0
Initital
Week 1
Week 2
Time
Fig. 3.4 Average cumulative wet weights of U. intestinalis, U. prolifera and U. lactuca with Aquasol full, mean ± se
(n=3). Different superscripts represent a significant difference (P<0.05).
Growth patterns of U. intestinalis were observed to be exponential and in the other two species it
appeared to be more linear (Figure 3.5).
6
Weight (grams)
5
4
U. lactuca
U. prolifera
3
U. intestinalis
2
1
0
Initial
Week 1
Week 2
Time
Fig. 3.5 Mean weekly weight gains for U. intestinalis, U. prolifera and U. lactuca. (n=3)
43
Discussion
The rapid growth of all three algal species seen in the current experiment is due to Ulva’s highly
efficient morphology. Its simple double-celled sheet-like thalli or single-celled hollow thalli
enables the species to respond rapidly to nutrient inputs with high rates of nutrient uptake and
growth. Based on the growth comparisons that were conducted in this work, either U. lactuca or
U. prolifera would provide maximum growth properties for optimum nutrient assimilation
capacities in a bioremediation system.
Opportunistic seaweeds such as Ulva have a high demand for nitrogen thus without sufficient
nitrogen concentrations growth is limited. Seaweeds can utilise nitrogen in several forms including
ammonia, nitrite, nitrate and as dissolved organic nitrogen, but ammonia is the most efficient form
for plant uptake (Neori 1996). Mariculture wastewater often high in nitrogen content, so the
culture of seaweed such as Ulva sp. in these waste streams is a logical step towards improved
productivity and reduced environmental harm from such activities. The importance of nitrogen for
Ulva growth is well demonstrated in the current experiment, both through the resultant colouration
and weight gains in the water type experiment. The nutrient concentration Aquasol full strength
(87 mg per litre) used in the current experiment appeared to be ideal with extremely high growth
rates recorded. This fertiliser has a range of balanced trace elements, compared with the Nanno
mix which is mainly nitrogen and phosphorus based. This more balanced nutrient availability may
explain the present results.
Without sufficient nutrients Ulva growth was observed to be
significantly less and a visual change in colouration was observed with a yellowing of the thallus.
Whilst nitrogen is one of the nutrients which has primary importance for plant growth, phosphorus
is also a major limiting nutrient in algal blooms. Dissolved forms of both of these major nutrients
44
are generally available in plentiful supply in the drainage water from sand worm beds used to filter
mariculture waste water (Palmer 2008). This is due to the bacterial breakdown of particulate
organic matter that is continuously deposited in the beds. Several other factors add to the
presumption that this worm bed drainage water will be very suitable for Ulva growth. These
include the reduction of microalgal densities it affords which increase light penetration into the
water column, and the removal of grazing herbivores like amphipods which can prevent
harvestable quantities of Ulva from developing in culture vessels.
Since researchers at BIRC are therefore interested in developing systems to grow Ulva sp. in this
worm bed drainage water, the present study sought to compare it with seawater. The results of this
comparison suggest that equal growth can be achieved in either medium as long as sufficient
nutrients are present. Although we had expected faster growth in the worm bed water, due to the
background availability of dissolved nutrients, other factors may have played a part in the results
and will need further investigation. These include the presence of other chemicals (eg: sulphur)
that may impede algal growth, or possibly competition with bacteria for ammonia. Because
amphipods were not present in the seawater growth medium, and were removed from seaweed
seed stock as it entered the laboratory with freshwater washing, this factor that is important in
outdoor situations played no part in these experiments.
Light is also well recognised as an important factor in Ulva growth. If Ulva is stocked at very high
densities its growth is impeded through self shading (Bartoli et al. 2005). In this regard, Jiménez
del Río et al. (1996) have recommended that Ulva should not be stocked at densities greater that
1.5 grams of wet weight per litre. Yet in the current experiment the stocking density was 5-20
grams per litre and growth was still maintained at a high level. This is likely because the Ulva in
this study was grown in the laboratory was under fairly intense lighting (10,000 lux), and because
45
the culture vessels were clear bowls illuminated unilaterally by a bank of fluorescent tubes. This
allowed Ulva throughout the entire culture to receive a high level of light.
3.5 Conclusions
Methods were developed which could produce rapid growth of Ulva in controlled laboratory
cultures; rapid growth was demonstrated under high nutrient concentrations and Aquasol full
strength was found to be an ideal fertiliser for optimal Ulva growth in the BIRC laboratory.
Although previous experiments had found that Ulva stocking density should not exceed 1.5 grams
per L in the current experiment it was established that much higher stocking rates could be used
because the unilateral banks of lights enabled Ulva throughout the culture vessels to receive
sufficient quantities of light. While it is likely that drainage water from a worm bed will provide
more favourable outdoor conditions for Ulva growth than untreated mariculture effluent, due to
greater light availability, more dissolved nutrients, and a lack of herbivorous grazers, no significant
growth differences were detected between seawater and worm bed water in the present work.
Future studies should be undertaken to explain why worm bed water was not seen to improve Ulva
growth under laboratory conditions.
46
Chapter Four
Sporulation Studies
4.1 Introduction
In the wild Ulva reproduction has an apparent correlation with tides and phases of the moon
(Smith 1947). In the laboratory, various studies have shown that Ulva can be successfully induced
into reproduction through a number of stressors including: raising or lowering the temperature;
increasing or decreasing light intensity; transferring to darkness; transferring from still to moving
water; increasing or lowering the concentration of nutrient compounds in the medium; and
renewing the medium. The last method combined with tearing of the thallus has been used as the
principal method for inducing reproduction in Ulva sp. in the laboratory in numerous previous
experiments (Nordby 1977). While extensive testing has been conducted on induced sporulation
in countries such as USA (Smith 1947; Lersten and Voth 1960; Niesenbaum 1988), Japan
(Hiraoka and Oka 1998; Dan et al. 2002; Hiraoka and Enomoto 2008) China (Lin et al. 2008),
Norway (Braten and Nordby 1973; Nordby 1974; Nordby 1977) England (McArthur and Moss
1979) and Denmark (Thiadens and Zeuthen 1967) currently there are no published studies on this
subject from southeast Queensland. Furthermore, many of the methods that have been used are not
very applicable to a commercial situation where large scale treatment will be necessary. The
following experiment therefore aimed to assess these aspects of induced sporulation of Ulva in
southeast Queensland. The methods tested were a combination of renewal of medium and
fragmentation, and considerations were given to the suitability’s of these methods for a
commercial application.
47
4.2 Materials and Methods
Well-developed thalli of U. prolifera, U. lactuca and U. intestinalis were collected from outdoor
stock cultures at BIRC on August 18, 2008. After collection, the thalli were washed three times in
fresh water to remove any potential grazers and then placed under laboratory conditions for two
weeks in the conditions previously determined for optimal vegetative growth.
After the two weeks of conditioning the algal material was assessed visually for reproduction and
any algal material that was currently in a reproductive state was excluded from the experiment. By
use of a razor blade the selected thalli were neatly and quickly cut into segments 5 x 5 mm for U.
lactuca, and 5 mm x natural widths (1 mm - 10 mm) for U. prolifera and U. intestinalis.
Segmented and whole thalli were then transferred into clean Petri dishes containing 30 mls of fresh
1 µm filtered seawater enriched with Aquasol (87 mg/L) using 1 g of Ulva per dish. Each Petri
dish was assigned a number from a table of random numbers and randomly placed onto a wire
bench situated inside a laboratory. The Petri dishes were housed at ambient temperature
(approximately 20°C) and illuminated unilaterally by banks of white fluorescent tubes attached to
the wall at 10,000 lux for 11 days. Photoperiod was run on a 12 h dark: 12 light cycle by an
automatic time clock.
Results of the experiment were judged by the number of gametes released from the thalli. As
sporulation tends to be expressed in the form of sharply divided areas of sporulating and nonsporulating cells, rather than intermingling of the two cell types, there is a distinctive colour
difference between green non-discharged areas of the thallus and colourless discharged areas
enabling each Petri dish to be visually graded (Nordby 1974). Gamete discharge was measured
using a 0 to 3 scale previously employed by Lersten and Voth (1960) (Table 4.1 and Figure 4.1).
48
Table 4.1 Ordered classification table used to assess sporulation in Ulva species (after Lersten and Voth, 1960).
Sporulation scale
No discharge
Discharge less than 1/3 of thallus cells
Discharge 1/3 of thallus cells
Discharge more than 2/3 of thallus cells
0
1
2
3
0 = No discharge
1 = Discharge less than 1/3
2 = Discharge 1/3
3 = Discharge more than 2/3
Fig. 4.1 Visual template designed in current experiment for ranking sporulation of Ulva in Petri dishes.
Each Petri dish was visually inspected at 12:00 noon at 24, 48, 72, 96, 168, 216 and 264 hrs after
the start of the experiment. The entire experiment was then repeated a further three times to see if
the level of sporulation varied throughout the month. Water parameters including temperature,
49
dissolved oxygen, salinity and pH were measured in three randomly selected experimental units at
the start of each new experiment. Observations of fouling were noted and photographs were taken
of the phytoplankton species which proliferated. The phytoplankton was then identified with the
use of the following guides: Foged (1987), Zheng Zhong (1989), Round et al. (1990), Hasle and
Syvertsen (1996) and Stafford (1999).
Data Analysis
A repeated measures ANOVA was conducted with the use of the statistical software GenStat (11th
edition) to analyse sporulation data (Rowell and 1976). A 5% level of statistical significance was
used, when data sets significantly violated the requirements for a repeated-measures design
ANOVA, the Greenhouse Geisser Epsilon correction was used to calculate a more conservative P
value for each F ratio (Greenhouse and Geisser 1959). The statistical package SPSS (14.0) was
used to determine if there were statistically significant differences between water parameters
between each week.
4.3 Results
4.3.1 Sporulation
All fragments were observed to be in a vegetative state prior to fragmentation (Figure 4.2, a).
During the culturing of both the fragmented and whole sections of Ulva cellular changes were
observed. Cells were seen to become displaced in preparation for gamete formation (Figure 4.2, b),
and this was soon either followed by the formation of gametangia within the cells (Figure 4.2, c) or
the cells returned to their initial position and remained vegetative.
50
a
b
10 µm
10 µm
dd
c
10 µm
ee
10 µm
f
10 µm
10 µm
Fig. 4.2 Thallus in vegetative state (a), Thallus with displaced chloroplasts (b) Thallus forming gametes (c) Thallus with fully formed
gametes (d), Thallus after release of gametes (e), gametes swimming in Petri Dish (f).
51
If formation of gametangia were to occur they were observed to move around within the cells then
exit through a pore in the cell leaving the cell empty and clear in colour (Figure 4.2 e). After the
release of the gametangia (Figure 4.2 f) gametes were distinctively recognised by their elliptic
shape and red eyespot, and they were observed swimming swiftly around the Petri dish propelled
by two flagella.
Data was found to significantly violate the requirement for repeated measures ANOVA as the
Greenhouse Geisser epsilon was greatly smaller than one (0.3240). To compensate for possible
effects of non-sphericity in the measurements compared this correction factor was applied to the
ANOVA (Table 4.2). The ANOVA with these corrections showed a statistically significant
interaction at the highest level Time, Species, Treatment and Week (P < 0.006).
Table 4.2. Summary of repeated measures ANOVA with three grouping factors (Species, Treatment, Week) and one
trial factor (Time) and their interaction effects on mean degree of sporulation in Ulva. A Greenhouse-Geisser epsilon
of 0.3240 was applied to the degrees of freedom for all time effects and interactions.
Source
Species
Treatment
Week
Species, Treatment
Species, Week
Treatment, Week
Species, Treatment, Week
Error
Time
Time, Species
Time, Treatment
Time, Week
Time, Species, Treatment
Time, Species, Week
Time, Treatment, Week
Time, Species, Treatment, Week
Error (Time)
Green house epsilon = 0.3240
df
2
1
3
2
6
3
6
48
Mean Square
7.30
4.01
3.21
2.89
1.51
1.87
0.99
0.67
F
10.83
5.96
4.76
4.30
2.25
2.78
1.47
34.58
p
<0.001*
0.0018*
0.005*
0.019*
0.054
0.051
0.207
6
12
6
18
12
36
18
36
288
0.52
0.07
0.08
0.16
0.07
0.05
0.06
0.04
0.01
27.02
3.93
4.24
8.26
3.83
2.60
3.56
2.54
<0.001*
0.006*
0.0018*
<0.001*
0.007*
0.005*
0.004*
0.006*
* denotes statically significant P<0.05
52
As an experimental seaweed U. intestinalis was far superior to the other two species,
demonstrating an average sporulation rate twice (0.48) that of U. lactuca (0.21) and more than six
times greater than that of U. prolifera (0.07). All three species showed a similar trend in beginning
sporulation 48 hrs after treatment. This tended to peak at 72 hrs post-treatment and stabilise after
72 hrs until the end of the experiment (264 hrs) (Figure 4.3).
0.7
0.6
Mean degree of sporulation
0.5
0.4
U. intestinalis
0.3
U. lactuca
0.2
U. prolifera
0.1
0
24
48
72
96
168
216
264
-0.1
-0.2
Time (hours)
Fig 4.3 Time profile of mean sporulation between species, mean ± se (n=12)
Fragmented thalli experienced a higher degree of sporulation over whole thalli. At the end of
experimentation (264 hrs) fragmented thalli had achieved on average more than twice the degree
of sporulation with an average of 0.41 in fragmented thalli and an average of only 0.19 in whole
thalli (Figure 4.4).
53
0.5
Mean degree of sporulation
0.4
0.3
Fragmented
0.2
Whole
0.1
0
24
48
72
96
168
216
264
-0.1
Time (hours)
Fig 4.4 Time profile of mean sporulation between treatments, mean ± se (n=36)
U. lactuca and U. intestinalis were both found to respond positively to fragmentation, with greater
sporulation rates in fragmented (0.42, 0.58) than whole (0.00, 0.38) thalli (Figure 4.5).
Fragmentation was not found to improve sporulation in U. prolifera, where the average degree of
sporulation was higher in whole thalli (0.11) than in fragmented thalli (0.02).
0.7
Average Sporulation
0.6
0.5
0.4
Whole
0.3
Fragmented
0.2
0.1
0
Species
3
U. intestinalis
2
U. lactuca
1
U. prolifera
0
4
Fig 4.5 Mean sporulation of fragmented and whole thalli in U. prolifera, U. lactuca and U. intestinalis 54
(n=84).
Sporulation was observed to significantly fluctuate between weeks (Figure 4.6). The highest
degree of sporulation was seen in week one with an average of 0.55 at the end of experimentation.
This was followed by week two (0.33) and then week four (0.27). Week three experienced on
average the least amount of sporulation with only a rate of only 0.05 at the end of experimentation.
Water qualities remained very stable during the experiment (Figure 4.7). A one-way ANOVA
performed showed that there was no significant difference between temperature (P> 0.05), salinity
(P> 0.05), dissolved oxygen (P> 0.05) and pH (P> 0.05) between the four weeks.
0.7
0.6
Mean degree of sporulation
0.5
0.4
Week 1
Week 2
0.3
Week 3
Week 4
0.2
0.1
0
24
48
72
96
168
216
264
-0.1
-0.2
Time (hours)
Fig 4.6 Time profile of mean sporulation between weeks, mean ± se (n=9)
55
40
Concentration
35
30
Temperature ºC
25
Salinity
20
Dissolved oxygen
mg/L
pH
15
10
5
0
0
1
2
3
4
5
Time
Fig 4.7 Water parameter values at the start of each week, mean (n=3)
4.3.2 Fouling
Culture dishes and or outdoor stock cultures were frequently observed to foul with marine
phytoplankton, isopods, amphipods, copepods and marine flies (Figure 4.8). In total 17 fouling
organisms were observed (See Appendix). Benthic epiphytes were observed to smother the thalli
of Ulva thereby reducing its photosynthetic capacity, whilst isopods and amphipods were observed
to feed directly on the tissue of the alga.
a
b
Fig. 4.8 Culture dish fouling (a) and outdoor culture stock fouling (b).
56
4.4 Discussion
The current experiment averaged zooid formation of less than 33%. Previous studies using similar
techniques have achieved levels of up to 90% zooid formation (Hiraoka and Enomoto 1998). The
low degree of sporulation achieved in the current experiment may be attributed to a number of
factors. One of these factors may be that the algal material was primarily gametophytic. Previous
work conducted by Hiraoka and Enomoto (1998) found differences in the degrees of sporulation
between fragmented sporophytes and gametophytes, with 90% of sporophytes and only 40% of
gametophytes becoming reproductively mature within three days. It is uncertain why this
difference occurs but its is believed to be due to the differences in the system of zooid formation as
sporophytes produce zoospores by meiotic and mitotic cell division, while gametophytes produce
gametes only by mitotic cell division.
Low sporulation levels could also be attributed to sporulation inhibiting substances released by
thalli when fragmented. The active chemicals are more readily released from fragmented thalli
than from whole plants (Neilsen and Nordby 1975). Keeping the fragment concentration low and
effectively reducing the concentration of inhibiting substance can improve generative divisions of
most cells. It is unknown if this affected the sporulation rates in the current experiment and this is
an important aspect for further studies, particularly if the process is to be undertaken at large scale.
Fragmentation generally improved sporulation rates with fragmented thalli on average having a
higher degree of sporulation than whole thalli. This result is consistent with work conducted using
U. mutabilis where fragmentation increased sporulation by 15.8 to 80% (Nordby 1977).
57
Formation of gametes in un-fragmented thalli is most likely a response due to the renewal of
medium. Previous studies by Thaidens and Zeuthen (1967) have shown that a renewal of the
culture medium can stimulate the formation of gametes in Ulva, presumably through changes in
water parameters including pH, temperature and salinity. Another factor which could have been
responsible for inducing sporulation in un-fragmented thalli is the fact that in order to obtain 1 g of
U. lactuca the thalli had to be torn. This small amount of damage to the thalli may have induced
some degree of sporulation in the un-fragmented experimental units.
The differences in the degrees of sporulation between U. prolifera, U. intestinalis and U. lactuca
may be due to the age of the plant material. Previous work conducted by Føyn (1955) found that
sporulation is age dependant with plants under two months old seldom successfully releasing
gametes and zoospores. In the current experiment it was believed that all of the algal material was
mature and well over two months old however the exact age of the algal material was unknown
and age may have contributed to some of the difference in the degree of sporulation between the
three species that were tested.
Another factor which has been found to result in a difference between sporulation is the number of
cellular layers. Previous work conducted by Nordby (1977) stated that double-layer fragments tend
to sporulate later than single-layered fragments. This finding was not observed in the current
experiment with both single-layered fragments U. prolifera and U. intestinalis reaching
reproductive maturity at the same time (48 hrs) as the double-layered fragments of U. lactuca.
No experimental cultures were found to release gametes within two days of treatment. At least two
days were needed for the completion of the maturation process in all three species. This result
coincides with previous studies which found that other species such as U. spinulosa release their
gametes after two to three days (Hiraoka et al. 2003). U. pertusa has been found to take four days
58
(Hiraoka and Enomoto 1998), U. mutabilis has taken two days (Nordby 1974, Nordby 1977), U.
prolifera has taken three days (Hiraoka and Oka 2008), and various other Ulva sp. have taken two
to five days (Lersten and Voth 1960).
Nordby (1977) reported that the optimum pH for sporulation lies between 8.0 and 8.5. Outside this
range the rate of sporulation was found to decrease. However earlier work conducted by Lersten
and Voth (1960) found that a pH value of between 6.5 and 7.5 gave the highest degree of
sporulation. In the current experiment the pH ranged from 7.67 to 8.01 at the time of transfer.
Sporulation was observed in the current experiment to generally be higher when the pH was above
7.50, and a lower pH level appeared to possibly have a negative effect with the lowest weekly
sporulation coinciding with the lowest pH. However these factors were not specifically tested.
Macroinvertebrate grazers (amphipods and isopods) were frequently observed in outdoor stock
cultures at BIRC. These cultures were approaching their useful life and were in need of renewal
when these observations were made, but the organisms detected were indicative of the particular
problems that would be faced by culturist’s at large scale. In more natural systems these
macroinvertebrate grazers have been found to have less detrimental effects of macroalgae growth.
Studies conducted by Kamermans et al. (2002) found that in the field the exclusion of grazing
amphipods and isopods did not stimulate Ulva growth, in fact growth rates were observed to be
higher in enclosures that allowed grazing. It was discovered that when epiphytic diatoms were
present the amphipod Gammarus locusta and isopod Idotea chelipes grazed on the epiphytes and
not on Ulva tissue. However, in natural systems wild macroinvertibrate populations are heavily
regulated by predation, and mainly by fish (Holmlund et al. 1990). Interestingly, amphipods were
noted to be difficult to find in tanks at BIRC where mullet and Ulva were both present, which
suggests that such a co-culture approach could be useful at large scale in the future.
59
In managed algal cultures there are normally no predators of amphipods and isopods and so their
population levels can build to very high unnatural levels with dire consequences for algal biomass.
In the present work they were observed to quickly overtake culturing bowls, and this was
minimised in experiments by thoroughly washing all of the seaweed biomass with freshwater to
kill these grazers prior to use. This appeared to be effective with almost all amphipods and isopods
dying within a few minutes of treatment.
Unfortunately, this freshwater treatment was not found to be so effective at removing
phytoplankton. Several laboratory cultures were observed to unintentionally become heavily
fouled with phytoplankton. For the purposes of the study, these were discarded, but it raised
additional concerns for similar problems that could be faced in commercial operations.
Phytoplankton and macroalgae are well known to compete for both nutrients and light. This
competition is the subject of many oceanographic studies, and an understanding of the processes at
work provides the basis for pond management activities in large scale aquaculture. A good
example of this relationship is provided by the annual model constructed to simulate the
competition between U. rigida and phytoplankton in the Venice lagoon. This research concluded
that while Ulva and phytoplankton constantly completed for available resources, Ulva is able to
successfully persist because it can grow efficiently at lower light intensities (Solidoro et al. 1997).
These results show that it is extremely important for all potential competitors and grazers to be
removed from cultures. This will also apply to cultures being seeded with algal spores and
germlings, since they are very delicate structures lacking the resistance mechanisms found in adult
material (Lubchenco 1983). Physical stress, competition and grazing can also suppress early
60
development stages (Vadas et al. 1992). It would therefore be sensible for future studies to build
stringent control measures into operating protocols.
As observed in the current experiment reproductive cells can be easily obtained using the current
fragmentation method. Swarmers and other induced propagules have been used for artificial
seeding in the past which has lead to a simplification of procedures (Dan et al. 1997). With this
method, reproduction is not restricted by season and potentially could be induced at any time under
laboratory conditions, as long as there are well preserved thalli of the algae present.
While sporulation was achieved in all three species through fragmentation the degree of
sporulation was unfortunately considered to be too low for commercial application. Further work
will therefore be needed to refining induction methods and achieve sporulation at larger scale.
4.5 Conclusions
Sporulation was effectively induced in all three species of Ulva at BIRC. Levels were generally
low and this could be attributed to a number of factors including the age of the algae material, the
fact the algal material was primarily gametophytes or the algae material could have leaked
sporulation inhibiting substances when fragmented. All of these factors have been previously
found to reduce sporulation. Whilst the level of sporulation achieved would not be suitable for
commercial application, this study provides a platform for future work in this area in the future.
To improve future sporulation rates it could be worth looking at water flow through the
fragmented material to reduce the amount of sporulation inhibiting substance, although the success
of this approach would be difficult to monitor experimentally. It is recommended that further
studies are conducted to improve the sporulation rates.
61
Chapter Five
Summary
5.1 Concluding summary
The objectives of the following study were to; identify all Ulva species naturally occurring at
BIRC (i), establish the most optimum conditions for growing Ulva in the laboratory (ii), test if
Ulva can be successfully induced into sporulation through a combination of fragmentation and
renewal of medium and if so quantify the degree of sporulation and assess if it is suitable for a
commercial application. This study achieved all of these objectives by identifying three species of
Ulva, U. lactuca, U. intestinalis and U. prolifera to occur at BIRC. Establishing that Aquasol was
the most favoured fertiliser for optimum growth and should be applied at a rate of 87 mg/L when
Ulva is stocked at a density of 5-10 grams per litre and finally by establishing that Ulva in
Southeast Queensland can be successfully induced into sporulation through a combination of
fragmentation and renewal of medium. It was concluded however that sporulation levels achieved
in the current experiment were too low for a commercial application and that future studies need to
be undertaken to improve this rate. Studies could possibly look into other known inducers
including dehydration, changes in salinity, nutrient, light and temperature as long as the methods
are able to be reproduced on a mass scale. While the current study didn’t achieve sporulation
levels desired for a commercial operation it did still play a vital role in contributing to improving
our understanding of inducing reproduction of Ulva within Southeast Queensland.
62
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Appendix
a
b
Zhong, Z. 1989. Marine planktology. China Ocean Press, Beijing, China, pp.454.
10 µm
c
10 µm
d
10 µm
e
10 µm
f
10 µm
10 µm
g
h
69
10 µm
10µm
10 µm
i
j
10 µm
10 µm
k
l
10 µm
10 µm
m
n
10 µm
o
10 µm
p
70
10 µm
10 µm
r
q
5 mm
s
5 mm
t
10 µm
10 µm
v
u
10 µm
Appendix 1. Licmophora sp. (a), Cylindrotheca (b), Unknown sp. (c), Pleurosigma sp. (d), Unknown sp. (e)
Oscillatoria sp. (f), Unknown sp. (g-p), Australian kelp fly pupae (q), Australian kelp fly (r), Unknown sp. (s)
Unknown sp. (t) ,Navicula sp. (u) and amphipod (v).
71